Major depressive disorder (MDD) is the one of the most common psychiatric diseases, and affects millions of people worldwide. Most drugs available to treat MDD target brain pathways involving monoaminergic neurotransmitters. Despite these therapies, as many as fifty percent of patients do not improve in response to existing therapies. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels mediate Ih, a current that is critical for controlling neuronal excitability and integration of synaptic inputs from other brain regions. HCN is tightly regulated by its auxiliary subunit, TRIP8b (tetratricopeptide repeat-containing Rab8b- interacting protein), which is responsible for trafficking HCN to the neuronal surface and is only expressed in the brain. Our lab and others have observed that loss of HCN channel function through either genetic deletion or by interfering with TRIP8b function leads to an antidepressant-like phenotype. Beyond its role in the brain, HCN is crucial for many functions outside the central nervous system, including rhythmogenesis in the heart. This crucial HCN function has confounded previous efforts to globally target HCN in MDD. We have developed a novel approach to discover new potential depression therapeutics by targeting HCN/TRIP8b interaction with small molecules. We have established a high throughput fluorescence polarization (FP) screening assay as well as secondary assays to identify HCN/TRIP8b inhibitors. We have validated our primary screening assay by testing a diverse library of small molecules and have further validated several of these hits in orthogonal assays to confirm target engagement and potency. Simultaneously, based on the known TRIP8b x-ray crystal structure, we have carried out a pilot in silico screen and verified several of the resulting hits in our biochemical assays. To identify inhibitors with improved potency and efficacy and find compounds more suitable for probe optimization, we propose to carry out expanded wet and in silico high- throughput screening utilizing our established primary and secondary assays. In specific aim 1, we will use our FP assay to test a library of 235,000 diverse small molecules for their ability to disrupt HCN/TRIP8b binding. We will also use our in silico protocols to screen the 18 million compound Zinc database and verify the resulting structures as inhibitors by the FP assay. In specific aim 2, we will assess the potency of our hits using a rational series of in vitro screening assays including fluorescence thermal shift (FTS), AlphaScreen, and glutathione S-transferase (GST) pulldown experiments. Finally, in specific aim 3, we propose to evaluate the functional activity of our hits in a series of cell-based assays including flow cytometry, immunocytochemistry, and electrophysiology. This proposal represents a novel approach to treat MDD and the small molecules that we discover will be broadly useful in developing new therapeutics for this unmet medical need as well as for probing the involvement of HCN channels and TRIP8b in depression.